PROJECT SUMMARY Cells and tissues are mechanosensitive. Many tissues, including the liver, are subjected to mechanical stresses and deformed at varying magnitudes and rates that can be altered in disease. When the mechanical properties of tissues change, as in fibrosis, or when stresses are abnormal, as with increased vascular pressures, cells are abnormally deformed, with resulting changes in cellular function that can initiate or augment disease. The design principles that determine tissue mechanics, however, are largely unknown. While the viscoelasticity of crosslinked semi-flexible polymer networks (ubiquitous in both the internal cytoskeleton and the extracellular matrix (ECM)) is generally assumed to dominate tissue mechanics, the mechanical responses of soft tissues and semiflexible polymer gels are the opposite of each other in many respects. Three-dimensional tissues stiffen in compression and mildly soften in extension, whereas semiflexible biopolymer networks soften in compression and stiffen in extension. Our overall goal is to use experimental and theoretical studies to determine how the presence of cells within fibrous networks can explain the viscoelastic response of intact tissues and thereby help explain how changes in the properties of either the matrix or the cells can alter the tissue mechanical properties that occur in disease. We propose to expand the knowledge generated during the first three years of funding of this proposal to further study the role of fibrous matrices in tissue and cell mechanics. Specifically, we will develop a more detailed understanding of the relationship between fibrous networks and cell and tissue responses to cell- generated or externally applied forces. This work will capitalize on our expertise and preliminary work on a model tissue, the normal and fibrotic liver, but the findings will be generally applicable to organs and soft tissues in the body. We will explore the role of complex fibrous networks in tissue behavior at various time and length scales, basing our work on the hypothesis that fibrous networks are critical determinants of tissue mechanics and of the behavior of cells within tissues. The three specific aims are to: 1) Define the role of the fibrous interstitial matrix of tissues in the mechano-responsiveness of real and model tissues and develop a multiaxial mechanical model of a simple tissue; 2) Determine the role of free and proteoglycan-bound GAGs in collagen fibrous networks and tissue mechanics; and 3) Define mechanisms that determine the plasticity (permanence) of tissue-scale matrix remodeling. For all aims, we will carry out both experimental and theoretical work, with the ultimate goal of understanding cell and tissue behavior in different physiologically-relevant matrix and mechanical environments. These studies will enable us to better understand the deleterious changes in tissue mechanics that are increasingly documented to contribute to (rather than simply result from) progression of diseases such as fibrosis. Ultimately, they may lead to innovative new treatments targeting specific mechanical features of diseased tissues.